Plant protein with repeated WD40 motifs, nucleic acid coding for said protein, and uses thereof

ABSTRACT

Isolated or purified nucleic acids, such as SEQ ID NO: 1, encoding a plant protein with repeated WD-40 motifs or fragments of such a protein that regulate plant differentiation or endoreplication. Vectors, host cells and plants comprising such nucleic acids, as well as antisense nucleic acids corresponding to such nucleic acids. Methods of using such nucleic acids, antisense nucleic acids, vectors, host cells, or proteins, for instance, for regulating plant differentiation or endoreplication.

This application is a national-stage filing under 35 U.S.C. §371 ofPCT/FR99/01342, filed Jun. 8, 1999. This application claims priorityunder 35 U.S.C. § 119 to France 98 07174, filed Jun. 8, 1998.

The invention relates to the cloning of genes involved in regulatingcell division in plants, and their uses.

Most plant organs develop after germination, through differentiationfrom the meristems. Prior to differentiation, the cell division cycleslows down and then stops in the meristems. Simultaneously, an increasein the size of the cells, and replication of the genome not accompaniedby mytosis, called “endoreplication”, are frequently observed.Endoreplication is a well known phenomenon during the development ofstorage tissue; KOWLES [Genome, 35, pp. 68–77, (1992)] thus mention aploidy of 6 C to 384 C during the development of the endosperm in maize.

The phenomena involved in the stoppage of cell division precedingdifferentiation play an essential role in plant development andontogeny. The mechanisms involved in these phenomena are still poorlyknown; it appears that the inhibition of the factor for promoting the Mphase, and the induction of the protein kinases of the S phase (GRAFI,Science, 269, pp. 1262–1264, (1995)] could be involved. However, nofactors directly involved in this mechanism have so far been identifiedin plants.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A represents a dendrogram of the family of proteins with repeatedWD40 motifs.

FIGS. 1B and 1C show that the CCS52Ms protein contains 7 domains withrepeated WD40 motifs.

FIG. 2 schematically shows the structure of the CCS52Ms protein.

FIGS. 3A and 3B show that expression of CCS52 causes the inhibition ofgrowth of S. pombe, which is accompanied by endoreplication (3B), butnot in control cells carrying the empty vector pREP1 (3A).

FIG. 3B shows the presence of nuclei ≧4 C.

FIG. 4 shows the positions of primers P55Bl, P55CL and P55Cr.

FIG. 5A shows the results of evaluation of the quantity of endogenousccs52Mt transcripts.

FIG. 5B shows the results of analysis by flow cytometry for petioles ofcontrol plants containing the gus gene, diploids (C_(2n)) or tetraploids(C_(4n)).

FIG. 6A shows the width of the petiole as function of the percentage ofpolyploid cells.

FIG. 6B shows the relative number of endoreplicated (>4 C) nuclei.

FIG. 6C shows the relative width of the petioles.

The inventors undertook the study of this mechanism with the aim ofdiscovering the means of controlling and of acting thereby on plantdevelopment and ontogenesis.

They chose, as a study model, the Rhizobium/leguminous plant symbioticsystem. In this system, the Nod factors, which are lipooligosaccharidein nature and which are produced by Rhizobium, constitute mitogenicsignals which locally induce the formation of a new meristem, from whichthe cells forming the root nodules become differentiated [TRUCHET,Nature, 351, pp. 670–673, (1991); YANG, Plant Cell, 6, pp. 1415–1426,(1994); SAVOURE, EMBO. J., 13, pp. 1093–1102, (1994)]. The nodulescomprise 3 main regions: an apical region, consisting of meristematiccells; an intermediate region for invasion or for differentiation(region II), where the infection of the cells by bacteria, as well asthe stoppage of cell division, accompanied by endoreplication and anincrease in the size of the cells, followed by their differentiation,take place; and a region for fixation (region III), consisting ofdifferentiated cells infected by bacteria, and where the fixation ofnitrogen takes place.

During this study, the inventors isolated, from lucerne (Medicagosativa) nodules, a gene, called hereinafter ccs52, which plays anessential role in the stoppage of the cell cycle and the induction ofendoreplication. Using a cDNA probe of the Medicago sativa ccs52 gene,they also isolated a homologous gene in Medicago truncatula.

The ccs52 genes of Medicago sativa (ccs52Ms), and of Medicago truncatula(ccs52Mt) encode a polypeptide of 475 amino acids having a theoreticalmolecular mass of 52 kDa. These polypeptides are called hereinafterCCS52Ms and CCS52Mt, respectively; the sequences of CCS52Ms and CCS52Mtdiffer by only 2 residues at positions 16 (R/G) and 141 (V/I).

These 2 proteins comprise repeated WD motifs, and may thus be attachedto the superfamily of proteins with repeated WD motifs.

The repeated WD motifs comprise about 40 amino acids containing a numberof conserved amino acids including the WD motif (Trp-Asp) which isfrequently situated at one end of the repeated motif [NEER et al.,Nature, 371, pp. 297–300, (1994)]. The members of this family regulatevarious functions, such as signal transduction, transcription, pre-mRNAsplicing, organization of the cytoskeleton, vesicular fusion or the cellcycle. Although the general structure is overall similar in all theproteins, the wide functional variety of repeated WD motifs suggeststhat these motifs have become differentiated and have becomefunctionally specialized. A functional homology is reflected in thenumber of repeated WD motifs, by a strong homology of the repeated WDmotifs with equivalent positions in various proteins, compared withother repeated motifs in the same proteins, and by a significantsimilarity of the C- and N-terminal ends.

Comparison of the sequence of CCS52Ms with the sequences of knownproteins, using the GENETICS COMPUTER GROUP GAP programme [parameters:gap weight: 1000; length weight: 0.100; average match: 0.540; averagemismatch: 0.396] reveals a high homology with the proteins containingrepeated WD40 motifs which are involved in the regulation of the cellcycle, and more specifically, with the Drosophila FZR proteins (57%identity), Saccharomyces cerevisiae HCT1 (46% identity), andSchizosaccharomyces pombe SRW1 (52% identity), which belong to the“fizzy-related” (FZR) family. Research carried out on databases ofsequences using the BLAST programme [ALTSCHUL et al. Nucleic Acids Res.25:3389–3402, (1997)] have also shown a strong homology of CCS52Ms withthe Drosophila FZR proteins (56% identity; 70% similarity), and theSchizosaccharomyces pombe SRW1 proteins (51% identity; 67% similarity)mentioned above, as well as with the product of the X. laevis fzr gene(58% identity; 73% similarity).

The FZR proteins induce the degradation of the mitotic cyclins and areinvolved in the transition between cell proliferation anddifferentiation. It has thus been shown in Drosophila that the fzr geneis expressed at the end of cell proliferation during embryogenesis. Theproduct of this gene causes a reduction in the mitotic cyclins, and isnecessary for the stoppage of cell proliferation and the start of theendocycles [SIGRIST and LEHNER, Cell, 90, pp. 671–681, (1997)]. InSaccharomyces cerevisiae, HCT1 is necessary for the proteolysis of themitotic cyclin, Clb2 [SCHWAB et al., Cell, 90, pp. 683–693, (1997)]. InSchizosaccharomyces pombe, the product of the swr1 gene controls thecell cycle and differentiation by negatively regulating the Cdc2/CDC13(cyclin of the mitotic type) complexes [YAMAGUCHI et al., Mol. Biol.Cell., 8, 2475–2486, (1997)]. The FZR proteins therefore have adifferent role from that of the other proteins with repeated WD motifs,which are involved in cell proliferation.

In plants, no protein of the FZR family had been described prior toCCS52Ms.

The existence of a gene encoding a protein with repeated WD40 motifs andits isolation from carrot cDNA have recently been described [LUO et al.,Plant Mol. Biol., 34, pp. 325–330, (1997)]. However, the product of thisgene exhibits a weaker homology (44% identity and 63% similarity on thesequence comparison carried out with the BLAST programme) with theCCS52Ms protein than the FZR proteins of invertebrates and of yeast;this carrot protein is related to the cdc20, p55 and fizzy proteins, andtherefore belongs to a subgroup of proteins with repeated WD40 motifsdistinct from the FZR subgroup.

The search for homologues of CCS52Ms in a database of the Arabidopsisthaliana genome has revealed a peptide sequence deduced from a genomicclone (AB005230) and exhibiting 64% identity with CCS52Ms, which showsthe existence of homologues of the ccs52Ms gene in other plants. Anotherpeptide sequence also deduced from a genomic clone of Arabidopsisthaliana (AL031018, published on 17 Sep. 1998) exhibits 80% identitywith CCS52Ms (44% identity and 63% similarity based on the sequencecomparison carried out with the BLAST programme).

FIG. 1A represents a dendrogram of the family of proteins with repeatedWD40 motifs, which shows that the CCS52 proteins form with the other FZRproteins a subfamily representing a branch which evolved separately fromthose respectively consisting of the CDC20, P55 and fizzy proteins.

FIGS. 1B and 1C represent alignment, carried out using the “PRETTYBOX”software, of Meedicago sativa CCS52 (MsCCS52) sequence (SEQ IS NO:2) andthe Drosophila FZY and FZR (DmFZY and DmFZR) sequence (SEQ ID NOS:7 and8), of the Saccharomyces cerevisiae HCT1 (ScHCT1) sequence (SEQ IDNO:9), the Schizosaccharomyces pombe SRW1 (SpSRW1) sequence (SEQ IDNO:10), the Arabidopsis thaliana FZY (AtFZY) sequence (SEQ ID NO:11) andthe 2 Arabidopsis thaliana polypeptides (AtCCS52a=peptide deduced fromAL31018 (SEQ ID NO:12), and AtCCS52B=peptide deduced from AB005230 (SEQID NO:13).

The CCS52Ms protein contains 7 domains with repeated WD40 motifs,situated in the central and C-terminal portions of the molecule (thelocation of these domains numbered from I to VII, is indicated in FIGS.1B and 1C, above the alignment of the sequences). These domains exhibitonly a slight homology with each other, hence it can be concluded thatthey represent sites for interaction with different proteins. The latterdomain (VII) comprises a potential binding site for the cyclins.

In the N-terminal portion of the CCS52Ms protein are localized a peptidesequence (DRFIPSR) which corresponds to a motif present in the FZRproteins as well as in other proteins with repeated WD40 motifs such ascdc20, p55 and fizzy, as well as a peptide sequence (AYTTLLRTALFG) whichcorresponds to a motif specific to the FZR family, absent from the otherproteins with repeated WD40 motifs (the location of these motifs, calledI and II respectively, is indicated in FIG. 1B above the alignment ofthe sequences).

Potential sites for phosphorylation with CDKs (cyclin-dependent kinases)are located in the N-terminal portion, at positions 43 (SPSR), 99(TPEK), 144 (SPVK), 154 (RSP) and 155 (SPYK), as well as in theC-terminal portion at position 454 (SPK), of CCS52Ms. The sites situatedat positions 43 and 144 are also present in other FZR proteins, whereasthe sites situated at positions 99, 154 and 155 appear more specific tothe CCS52 proteins of plants; the C-terminal site at position 454 alsoappears to be specific to the CCS52 proteins of plants.

A sequence of 15 amino acids RDNSPPPEPSPESLR starting at residue 16, andcorresponding to a protein degradation motif PEST is also present in theN-terminal portion of CCS52Ms. This motif probably makes it possible,through the degradation of CCS52, to regulate its interactions withother proteins.

The structure of the CCS52Ms protein is schematically represented inFIG. 2, in which the position of the WD40 motifs, of the phosphorylationsites (P), of the PEST motif, and of the I and II motifs, are indicated.

The sequence of the Medicago sativa cDNA cloned by the inventors isrepresented in the sequence listing in the annexe under the number SEQID NO:1; the sequence of the corresponding CCS52Ms protein isrepresented under the number SEQ ID NO:2.

The untranslated 3′ region of the transcript of this DNA comprises 2AUUUA sequences, which correspond to sequences for instability of themRNA, and may therefore play a role in regulating the quantity oftranscripts of ccs52.

The inventors searched for the presence of homologues of ccs52Ms bySouthern transfer, in diploid and tetraploid species of Medicago, aswell as in other plants, in particular tobacco, tomato, potato, soya,wheat and rice: in all cases, several bands were detected, whichindicates that ccs52 indeed represents a family of plant genes which isrelated to the fzr family.

The inventors studied in vivo the activity of the CCS52Ms protein andshowed that it was involved in regulating cell differentiation, and inpromoting endoreplication. In particular, the expression of the CCS52Msprotein in transgenic plants induces therein an increase inendoreplication and in the level of ploidy of the cells of plants. Thiseffect could be the consequence of a blocking of mitosis by theactivation of the degradation of the mitotic cyclins, which would bringabout conversion of the mitotic cycles to endocycles consisting of theG1-S-G2 phases. The result of the repetition of the endocycles is theamplification of the genome and the increase in ploidy, correlated withan increase in cell volume.

The subject of the present invention is a plant protein with repeatedWD40 motifs, called CCS52, characterized in that it belongs to the FZRsubfamily.

According to a preferred embodiment of the present invention, the saidplant protein exhibits at least 45%, and preferably at least 55%identity with the polypeptide having the sequence SEQ ID NO:2 or atleast 60% and preferably at least 70% similarity with the polypeptidehaving the sequence SEQ ID NO:2.

The present invention includes in particular the CCS52Ms protein, itsisoforms, as well as the autologous proteins of Medicago and theorthologous proteins of other plants, which may be attached to thefamily of FZR proteins.

The invention also includes proteins derived from the CCS52 proteins byaddition, deletion or substitution of one or more amino acids or of oneor more amino acid sequences; this may include for example proteins inwhich modifications have been made outside the functional regions, oralternatively proteins in which modifications have been made in order tomodify their activity, for example proteins stabilized by deletion ofthe PEST motif.

The subject of the present invention is also a purified nucleic acidfragment, characterized in that it comprises all or part of a sequenceencoding a CCS52 protein, as defined above, or its complementarysequence. In this context, the present invention includes in particularthe cDNAs and the genomic DNAs of the CCS52 proteins.

Nucleic acid fragments in accordance with the present invention can beeasily identified and cloned by screening plant cDNA or genomic DNAlibraries with the aid of oligonucleotides derived from the ccs52Mssequence, and in particular oligonucleotides derived from the regions ofthis sequence which are specific to the FZR proteins, and in particularthe CCS52 proteins.

The CCS52 proteins may be produced, in particular, by expressing thesenucleic acid sequences in host cells.

The subject of the present invention is also the use of a CCS52 protein,as defined above, or of a nucleic acid sequence encoding all or part ofthe said protein, or of its complementary sequence, for regulating thedifferentiation and the proliferation of plant cells.

The subject of the present invention is also the use of a protein of theFZR subfamily or of a nucleic acid sequence encoding all or part of thesaid protein, or of its complementary sequence, for regulating thedifferentiation and the proliferation of plant cells.

There may be mentioned, among such proteins, the drosophila FZR proteinor the yeast FZR protein.

The modification of the expression and/or of the activity of CCS52proteins in plant cells makes it possible to modify the cell cycle, bypromoting either proliferation or differentiation, and to thus controlthe development process, in order to obtain, for example, stimulation ofsomatic embryogenesis, to increase in vitro regeneration of plants fromcalli, by increasing conversion to embryos, or to promote thedevelopment of certain organs, for example to increase the productivityof storage tissues by increasing their endoploidy.

It is possible in particular to use the cDNA sequences of CCS52 proteinsor of portions of these cDNA sequences, or of their sense or antisensetranscripts; this may be for example the entire sequence encoding aCCS52Ms protein, or a portion of this coding sequence, and/or all orpart of the untranslated 5′ and 3′ regions. These sequences may be usedin the sense orientation, or if it is desired to inhibit the expressionof the CCS52Ms protein in a plant or in a tissue or organ thereof, inantisense orientation.

The present invention also includes recombinant DNA constructscontaining at least one nucleic acid sequence in accordance with theinvention.

Generally, the said nucleic acid sequence will be placed undertranscriptional control of an appropriate promoter.

Advantageously, it will thus be possible to use a strong promoter inorder to increase, in the host cells, the levels of expression of theCCS52 protein; this may include an inducible promoter or a constitutivepromoter, a ubiquitous promoter, or a tissue-specific promoter.

The use of inducible promoters makes it possible to obtain blocking ofmitosis, and the induction of endoreplication at the desired moment. Theuse of tissue-specific promoters makes it possible to target the actionof the CCS52 protein at certain tissues and organs (for example storagetissues).

By way of examples of strong promoters which can be used in the contextof the present invention, there may be mentioned: the CaMV35S [BENFLY etal., Science, 250, pp. 959–966, (1990)], the 35S promoter; theAgrobacterium tumefaciens T-DNA promoters: nopaline synthase, octopinesynthase, mannopine synthase, 1′, 2′ [SANDERS et al., Nucleic Acid Res.,15, pp. 1543–1558, (1987); HOOYKAAS and SCHILPEROORT, Plant. Mol. Biol.,19, pp. 15–38, (1992)].

By way of examples of inducible promoters which can be used in thecontext of the present invention, there may be mentioned: the promoterinducible by tetracycline [WEINMANN et al., Plant J., 5, pp. 559–569,(1994)]; the promoter inducible by copper [METT et al., Transgenic Res.,5, pp. 105–113, (1996)]; the promoter inducible by glucocorticoids[AOYAMA and CHUA, Plant. J., 11, pp. 605–612, (1997)].

By way of examples of tissue-specific promoters which can be used in thecontext of the present invention, there may be mentioned: theendosperm-specific promoter [OPSAHL-FERSTAD et al., Plant J., 12, pp.235–246, (1997); DOAN et al., Plant Mol. Biol., 31, pp. 877–886, (1996);the nodule-specific promoters (enod12A/B or leghaemoglobin) [TRINH etal., Plant Cell Reports, (17, pp. 345–355, (1998); VIJN et al., PlantMol. Biol., 28, pp. 1103–1110, (1995)] or early promoters inducible bythe Nod factor and late promoters (promoter of cyclin D or of latenodulins (leghaemoglobin type) and promoters regulated by hormones, suchas parA/B [TAKAHASHI et al., Proc. Natl. Acad. Sci, USA, 87, pp.8013–8016, (1990)], GH3 [LIU et al., Plant Cell, 6, pp. 645–657,(1994)].

The invention includes in particular recombinant vectors carrying atleast one insert containing a DNA fragment in accordance with theinvention. These vectors can be used for transforming host cells.

The subject of the invention is also cells and pluricellular organismstransformed with at least one DNA sequence in accordance with theinvention; this includes in particular plant cells or plants.

The present invention will be understood more clearly with the aid ofthe additional description which follows, and which refers tononlimiting examples illustrating the identification, cloning andexpression of the CCS52Ms gene.

EXAMPLE 1 Cloning and Sequencing of a CCS52Ms cDNA

A cDNA clone of CCS52Ms was obtained by differential screening from acDNA library of Medicago sativa nodules, highly stimulated duringnodular organogenesis.

The following protocol was used:

The cDNA of M. sativa ccs52Ms was isolated by the DD-RT-PCR(Differential Display RT-PCR) technique [LIANG and PARDEE, Science, 257,pp. 967–971, (1992)], using the RNAimage® kits (GENHUNTER CORPORATION).The RNA samples are isolated from the root region sensitive to the Nodfactor of young M. sativa plants (growth in a nitrate-limited medium),in the absence of bacteria or inoculated with Nod⁺ (EK1433) or Nod⁻(EK133) strains of R. meliloti for 4 days. The DD-RT-PCR ccs52Msfragment, exhibiting an increase in the expression of the nodules, iscloned into the cloning vector PCT-TRAP (GENHUNTER CORPORATION) and usedas a probe for the isolation of complete clones from a cDNA library ofnodules of M. sativa sp. varia A2, constructed in λ-ZAP (STRATAGENE)(CRESPI et al., EMBO J., 1994, 13, 5099–5112).

Seven cDNA clones, obtained from 2.10⁵ phages, represent 2 types of cDNAdiffering from each other only in the 4 amino acids (16R-G, 17D-N,33S-N, 52R-G) and the length of the 3′UTR fragment. A 99% identity forthe clones, at the level of the amino acid sequence, suggests that theyrepresent allels of the same gene in allogamous tetraploid M. sativa.

The sequencing of the ccs52Ms cDNA is carried out with the PERKIN-ELMERABIprism system.

The genomic clones ccs52Ms and ccs52Mt are isolated from genomiclibraries of M. sativa cv. Nagyszénasi and M. trucatula ecotype GHOR,using the ccs52Ms cDNA as hybridization probe. These genomic librariesare constructed by partial digestion of the genomic DNA with therestriction enzyme MboI and the cloning of the DNA fragments having asize of between 15 and 20 Kb into the BamHI site of λ-EMBL4.

EXAMPLE 2 Identification of the Family of the CCS52MS Gene in Medicagoand its Expression in Various Plant Organs

The existence of multiple copies of the ccs52 gene is tested for byhybridization of the Southern type in tetraploid cultivars of M. sativaNagyszénasi and Cardinal and in autogamous diploid M. truncatula, amodel plant in research on vegetables.

The plant DNA is isolated from young leaves, using the NUCLEON PHYTOPUREDNA extraction kit (AMERSHAM).

The DNA samples are digested with EcoRI and transferred onto BIOTRANSnylon membrane (+) (ICN).

The Southern hybridization is carried out in accordance withconventional protocols [(SAMBROOK, Molecular Cloning: A LaboratoryManual 2^(nd) edn., Cold Spring Harbor Laboratory Press, New York,(1989); AUSUBEL, Current Protocols in Molecular Biology, (1989)], understringent conditions at 65° C. (hybridization in CG buffer; washing:2×SSC, 0.1% SDS for twice 15 min, then 0.5×SSC, 0.1% SDS for twice 30min).

The expression of ccs52Ms is studied by Northern analysis.

Total RNA is isolated from various organs of M. sativa cultivar Sitel:

from the roots, inoculated for 4 days with the R. meliloti Nod⁻ mutant(EK133) and with the strain overproducing Nod factors (EK1433);

from the nodules, 12, 19, 23 and 30 days after infection with R.meliloti, and

from the stems, hypocotyls, leaves, buds, flowers, roots of plants whichare 3 days old, 7 days old, roots deprived of nitrogen and which do nothave root tips, roots which are 7 days old, without root tips, placed inculture in the presence of nitrate, spontaneous nodules developed in theabsence of R. meliloti, and root tips or a culture of cells of M. sativasp. varia A2.

100 mg of each of the organs tested, collected under liquid nitrogen,are used for the extraction of the RNA (RNEASY PLANT, QUIAGEN).

The RNA is loaded (10 μg per lane) onto a denaturing gel (formaldehyde)[SAMBROOK, Molecular Cloning: A Laboratory Manual 2^(nd) edn., ColdSpring Harbor Laboratory Press, New York, (1989)].

The DNA is transferred into a 10×SSC transfer solution [CHOMCZYNSKI etal., Analytical Biochemistry, 221, pp. 303–305, (1994)].

Both in the case of the Southern hybridization and in the case of theNorthern hybridization, the ccs52Ms cDNA fragment is labelled with[α³²P]dCTP (kit MEGAPRIM, AMERSHAM). Hybridization with the Msc27 probeserves as control for the loading of the RNA (SAVOURE et al., EMBO J.,13, pp. 1093–1102, (1994)].

The results of the Southern transfer show that the probe hybridizes withvarious EcoRI fragments of the genomic DNA of M. sativa or M.truncatula, which indicates that ccs52Ms represents, in Medicago, amultigene family.

The results of the Northern transfer obtained with the total RNA ofroots inoculated with the Nod⁻ EK133 mutant of R. meliloti, or with theEK1433 strain overproducing Nod factors and with the RNA extracted fromthe nodules, 12, 19, 23 and 30 days after infection with R. melilotishow that only a small quantity of transcripts is observed in the totalRNA of the roots, which reflects the small proportion of cells involvedin the organogenesis of the nodules compared with the total number ofcells of the roots. By contrast, in the nodules of different ages, ahigh level of transcription is observed, which reflects the persistenceof the apical meristems and of the regions for differentiation.

The results of the Northern transfer which are obtained with the totalRNAs of: 1: culture of cells of M. sativa sp. varia A2, 2: stems, 3:hypocotyls, 4: leaves, 5: flower buds, 6: flowers, 7: roots of shootswhich are 3 days old, 8: roots of shoots which are 7 days old, deprivedof nitrogen, lacking ends, 9: root tips which are 7 days old, culturedin the presence of nitrates, lacking ends, 10: spontaneous nodulesdeveloped in the absence of R. melioti, 11: nitrogen-fixing nodules, 12:ends of root tips, show that the expression of ccs52Ms is not limited tothe nodules, although this organ is that which contains the highestlevel of transcripts.

These transcripts are indeed present in variable quantities practicallyin all the organs, which indicates that this protein is involved in thedevelopment of each of them. Apart from the nodules, the level oftranscription is also high in young shoots, and, in cell cultures, wherea smaller sized mRNA is in addition detected which may correspond eitherto a different polyadenylation, or to the expression of a homologouscopy of the gene.

Analyses were also carried out by in situ hybridization, and show thatthe mRNA of ccs52Ms is located mainly in the region for differentiation,and in particular at the interface between regions II and III of thenodule, which are regions where differentiation is the most active.

In parallel, expression of the G1 and mitotic type cyclins as well as ofthe H3 histone specific to the S phase is observed in the same regions.

This indicates that CCS52Ms is involved in the regulation of the cellcycle, probably in a manner similar to its yeast and drosophilahomologues, that is to say by means of the proteolysis of mitoticcyclins, which inhibits mitosis and induces endoreplication cycles.

EXAMPLE 3 Expression of CCS52MS in Schizosaccharomyces Pombe

The expression of CCS52Ms was studied in S. pombe in which a functionalhomologue (SRW1) was recently described (YAMAGUCHI, publication citedabove). The gene encoding CCS52Ms was cloned into the plasmid into pREP1under the control of the nmt1 promoter which is repressible by thiamine.

The cDNA of ccs52Ms obtained after cleavage of λ-ZAP (STRATAGENE) isdigested with AgeI and partially with EcoRV. The AgeI-EcoRV fragment of1.6 kb representing the coding region, with the exception of the first 4codons, is cloned into a vector SKII BLUESCRIPT (STRATAGENE) digestedwith XmaI (compatible with AgeI) and EcoRV. From this plasmid (pSK52B),the cDNA of ccs52Ms is cut by BamHI-EcoRV digestion and cloned into theBamHI-SmaI sites of the plasmid pREP1 [MAUNDRELL et al., Gene, 123, pp.127–30, (1993)]. To generate an open reading frame in phase with the ATGcodon for translation present in the vector under the control of thenmtI promoter, the DNA is digested with BamHI and the 5′ end iscompleted in the presence of the Klenow enzyme and of dNTPs. Thereligation of the blunt ends causes correct fusion, also verified bysequencing. This plasmid, called pREP52, is used to transform competentS. pombe SP-Q01 cells and the transformants are selected on EMM-thiamineagar plates, using the ESP kit (STRATAGENE). The vectors pREP1[MAUNDRELL et al., Gene, 123, pp. 127–30, (1993)] and pESP1 (STRATAGENE)are used as negative controls; the positive control consists of srw1cloned into pREP1 [YAMAGUSHI et al., Mol. Biol. Cell., 8, pp. 2475–2486,(1997)].

The transformants of S. pombe SP-Q01 are cultured in 2 ml of 5 μMEMM-thiamine medium for 32 h at 30° C. The cells are washed twice with10 ml of sterile water and resuspended in 5 ml of EMM medium. Thecellular suspensions are divided into two halves: 2.5 ml are culturedwith thiamine and 2.5 ml are cultured without thiamine, at 30° C.Culture aliquots are collected after 16 h and 24 h of culture and fixedwith ethanol, stained with DAPI or with propidium iodide for analysis byflow cytometry and by microscopy [BEACH et al., Curr. Genet., 10, pp.297–311, 1985)].

In the presence of thiamine, the expression of CCS52Ms is repressed andnormal growth is observed.

In the absence of thiamine, the expression of CCS52Ms causes theinhibition of the growth of S. pombe, which is accompanied byendoreplication as illustrated in FIG. 3B, which shows the presence ofnuclei ≧4 C, which is not observed in the control cells of S. pombe,carrying the empty vector pREP1 (FIG. 3A).

The morphology of the cells is also modified by the expression ofCCS52Ms. A lengthening of the cells and an increase in the size of thenuclei are observed, which are identical to those observed during theexpression of SRW1 [YAMAGUSHI et al., Mol. Biol. Cell., 8, pp.2475–2486, (1997)], whereas no morphological change is observed when S.pombe carries only the vector pREP1.

In S. pombe, SRW1 is essential for the degradation of the mitotic cyclinCDC13. To verify if CCS52 acts in the same manner, the quantity of theCDC13 was evaluated in cultures of a strain (SY1) of S. pombe, carryinga deletion in the srw1 gene, and not degrading CDC13.

The total proteins obtained from cultures of SY1 transformed with pREP1(control) or with pREP1-ccs52 was analysed by Western transfer, andvisualized with the aid of anti-CDC13 antibodies.

In parallel, the expression of CDC2 kinase and that of α-tubulin wereevaluated by visualization with the aid of anti-PSTAIR andanti-α-tubulin antibodies, respectively.

The results obtained show a very high reduction in CDC13 in the cellstransformed with pREP1-ccs52 compared with the control cells. Bycontrast, there is no variation in CDC2 and in α-tubulin.

These results confirm that CCS52 is a functional equivalent of SRW1.

EXAMPLE 4 Production of Transgenic Plants Transformed with the CCS52MSGene

1. Expression of an Antisense Transcript and its Action on the Level ofPloidy of Medicago truncatula.

In a first instance, the level of ploidy of various organs of Medicagotruncatula (plant which is naturally diploid) was determined, by flowcytometry, in nontransformed plants.

The technique used is the following:

The nuclear DNA of freshly harvested plants is analysed by flowcytometry (EPICS V, Coulter), in accordance with the method of BROWN etal., (A laboratory guide for Cellular and Molecular plant Biology, 1991,326–345, ed. Negrutiu et al., Birkhaüser, Basel), modified such that thenuclei are stained with DAPI at a final concentration of 5 μg/ml. Thenuclear buffer I is used at 1% Triton X-100 for the nodules.

In young shoots, a quantity of DNA from 2 C to 8 C is found in the rootand the cotyledon, whereas the hypocotyl also contains nuclei at 16 C.In adult plants, the leaves are diploid, containing 95% of nuclei at 2 Cand 5% of nuclei at 4 C. In the petioles and the nodules, nuclei from 2C to 32 C were detected. However, the petiole contains predominantlynuclei at 2 C, whereas the nodules contain predominantly nuclei at 4 C.

An SstI-PvuII fragment of 1.2 kb containing ¾ of the coding sequence ofccs52Ms, was placed in antisense orientation under the control of the35S promoter, in a binary vector obtained from the vector pGPTV-BAR,carrying the bar gene for resistance to the herbicide BASTA asselectable marker, and multiple cloning sites. This construct isobtained by inserting the 35S promoter into a HindIII-XbaI fragment(obtained from pBI121, CLONTECH), into the HindIII-XbaI sites of thevector pGPTV-BAR. The uidA gene is then removed from the plasmidpGPTV-BAR by XbaI-SstI digestion at the level of the multiple cloningsite.

To obtain the antisense construct of ccs52Ms, the SstI-PvuII fragment of1.2 kb is cloned into the SmaI-SstI sites of the binary vector thusobtained.

These plasmids as well as a control plasmid, containing the gus geneinstead of the antisense ccs52 construct were introduced intoAgrobacterium tumefaciens (EHA105) by electroporation and used totransform Medicago truncatula R108-1 according to the protocol describedby HOFFMANN et al. [Mol. Plant Microbe Interaction, 10, pp. 307–315,(1997)]; TRINH ET AL. [Plant Cell Reports, 17, pp. 345–355, (1998)].

The level of ploidy of the transgenic plants obtained was analysed, asdescribed above and the level of endogenous transcripts was evaluated byRT-PCR. To differentiate the endogenous transcripts of ccs52Mt from theantisense transcripts, the pair of primers P55CL/P55CR is used for theendogenous transcripts and the pair of primers P55BL/P55CR for theantisense transcripts.

P55BL: TTTGGGGGTTGATGATTGTG SEQ ID NO:3

P55CL: CTCTCTACCGTTCTATCTCTTGGGA SEQ ID NO:4

P5CR: GGTAAAGATGCTACTTTGGTGGTGT SEQ ID NO:5

The position of these primers is schematically represented in FIG. 4.

FIG. 5A shows the results of evaluation of the quantity of endogenousccs52Mt transcripts:

-   -   by RT-PCR (□) in the transgenic lines A1, A3, A4, A7 and A32 and        in the control plants containing the gus gene (C_(2n)), and    -   by Northern transfer (▪) in A4 and C_(2n) plants.

The results of analysis by flow cytometry are illustrated by FIG. 5B,for the petioles of control plants containing the gus gene, diploids(C_(2n)) or tetraploids (C_(4n)), and of plants of the A4 line.

Out of 38 regenerated transgenic plants, 3 (A4, A7 and A32) showed asignificantly reduced endoploidy, and in particular the plant A4. It isalso in this line that the level of expression of the endogenoustranscripts of ccs52Ms is the lowest, as shown in FIG. 5B. The fact thata reduction in endoploidy was never observed before in other transgenicplants and are not observed in the control plants makes it possible toattribute this phenomenon to the impairment of the expression ofCCS52Ms, and not to a secondary effect of transgenesis.

In addition, the plant A4 produces a quantity of seeds significantlyless than that of the control plants. Moreover, it forms fewer sidebranches, and develops only 2 nodules at the level of the roots, insteadof the 50 nodules on average developed by the control plants culturedunder the same conditions.

The impact of the partial suppression of the expression of ccs52 on thedevelopment of the plant organs was also determined. For this purpose,the width of the petioles was measured and correlated with thepercentages of endoreplicated nuclei (>4 C), in the T1 generationderived from the A4 line and in the C_(2n) and C_(4n) control plants.

The results are illustrated in FIG. 6. FIG. 6A which represents thewidth of the petiole as a function of the percentage of polyploid cellsshows that, in the C_(2n) control plants (18 plants), the width of thepetioles varies in correlation with the number of diploid cells. In theplants derived from A4 (36 plants), a more reduced variation in the sizeof the petioles and a lower percentage of polyploid cells are observed,which indicates that the degree of endoploidy can directly affect thefinal size of the plant organs.

12 of the 36 T1 plants derived from A4 contain less than 6% ofendoreplicated nuclei (>4 C) in their petioles (FIG. 6B). These plants[A4 (s)] were grouped together and analysed separately from the rest ofthe A4 T1 plants [A4(w)] which exhibit less substantial phenotypicimpairments.

FIG. 6C shows that the width of the petioles in A4(w) plants iscomparable to that of the diploid C_(2n) control plants; by contrast,the width of the petioles in the A4(s) plants is significantly less thanthat of the diploid C_(2n) control plants and the width of the petiolesin the tetraploid C_(4n) control plants is significantly greater thanthat observed in the diploid plants.

The size of the leaves (which do not contain endoreplicated cells andwhose endoploidy is not therefore affected by the level of expression ofCCS52) was also measured. In this case, no significant difference isobserved between the A4(w) plants, the A4(s) plants and the diploidC_(2n) control plants; by contrast, the size of the leaves issignificantly larger in the tetraploid C_(4n) control plants.

These results show that endoploidy affects the size of the plant organs,and that the modification of the expression of CCS52 acts at this levelthrough a modification of the endoploidy.

2. Expression of the CCS52Ms Protein in Transgenic Plants.

Expression vectors containing the ccs5Ms gene under the control of the35S promoter, as well as expression vectors containing the ccs52Ms gene,under the control of a tissue-specific promoter, were constructedaccording to the following protocol:

For the tissue-specific expression of CCS52Ms, the cDNA is placed underthe control of the enod12AMs and Srglb3 promoters described by TRINH etal. [Plant Cell Reports, 17, pp. 345–355, (1998)], using as a vectorpISV-BMCS, a derivative of pISV2301, and, instead of the completeenod12AMs promoter, only one 0.3 kb fragment thereof, considered to besufficient for a nodule-specific expression [VIJN et al., Plant Mol.Biol., 28, pp. 1103–1110, (1995)].

Construction of pISV-BMCS: pISV2301 is digested with HindIII and SstI inorder to eliminate the sequence of the 2X35S-AMV promoter, which isreplaced by the following double-standed BMCS oligonucleotide:

AGCTTCCCGGGGGAGCTCTAGACTCGAGCAGCT

AGGCCCCTCGAGATCTGAGCTCG (SEQ ID NO:6).

This oligonucleotide contains the SmaI, SstI, XbaI and XhoI sites.

pISV-BMCS12A is constructed by cloning into pISV-BMCS of a fragment ofthe 0.3 kb of the endo12AMs promoter, obtained from the plasmid pPR89[BAUER et al., Plant J., 10, pp. 91–105, (1996)].

pISV-BMCS-LB3 is constructed by digestion of pISV-BMCS with HindIII-SstIand cloning of a HindIII-SstI fragment containing the leghaemoglobinpromoter of Sesbania rostrata from pLP32 [TRINH et al., Plant CellReports, 17, pp. 345–355, (1998)].

These vectors were used to transform Medicago truncatulata according tothe protocol described above for the antisense sequences.

During the regeneration of the transgenic plants, a significantlygreater conversion of the calli to embryos is observed in plantstransformed with the constructs expressing the ccs52Ms gene, than inplants transformed with the control construct, which indicates apositive effect of CCS52Ms on somatic embryogenesis.

1. An isolated or purified nucleic acid comprising: (a) a polynucleotidesequence encoding the polypeptide of SEQ ID NO: 2, or (b) apolynucleotide sequence encoding a polypeptide that inhibits mitosis andinduces endoreplication, wherein said polynucleotide hybridizes understringent conditions to the full-length complement of the coding portionof SEQ ID NO: 1, wherein said stringent conditions comprise at 65° C.:hybridization in CG buffer, washing in 2×SSC, 0.1% SDS for twice 15minutes, then washing in 0.5×SSC, 0.1% SDS for twice 30 minutes.
 2. Anisolated or purified nucleic acid which comprises a polynucleotidesequence encoding the polypeptide of SEQ ID NO:
 2. 3. The nucleic acidof claim 2 that comprises polynucleotides 182 to 1609 of SEQ ID NO: 1.4. The nucleic acid of claim 1, which comprises a polynucleotidesequence encoding a polypeptide that inhibits mitosis and inducesendoreplication, wherein said polynucleotide hybridizes under stringentconditions to the full-length complement of the coding portion of SEQ IDNO: 1, wherein said stringent conditions comprise at 65° C.:hybridization in CG buffer, washing in 2×SSC, 0.1% SDS for twice 15minutes, then washing in 0.5×SSC, 0.1% SDS for twice 30 minutes.
 5. Thenucleic acid of claim 1 that is isolated from a plant.
 6. A vectorcomprising the nucleic acid of claim
 1. 7. The vector of claim 6,wherein said nucleic acid is placed under the control of a promoter. 8.The vector of claim 7, wherein said promoter is an inducible promoter, aconstitutive promoter, a tissue-specific promoter or an ubiquitouspromoter.
 9. The vector of claim 7, wherein said promoter is aninducible promoter.
 10. The vector of claim 7, wherein said promoter isa tissue specific promoter.
 11. A host cell comprising the nucleic acidof claim
 1. 12. A plant cell that comprises the nucleic acid of claim 1.13. A transgenic plant comprising the nucleic acid of claim
 1. 14. Anisolated or purified nucleic acid comprising: (a) a polynucleotidesequence which comprises the full complement of the polynucleotidesequence of SEQ ID NO: 1, or (b) a polynucleotide sequence whichcomprises the full complement of the 1.2 kb SstI-PvuII fragment of SEQID NO:
 1. 15. A vector comprising the nucleic acid of claim
 14. 16. Thevector of claim 15, wherein said nucleic acid is placed under thecontrol of a promoter.
 17. The vector of claim 15, wherein said promoteris an inducible promoter, a constitutive promoter, a tissue-specificpromoter or an ubiquitous promoter.
 18. The vector of claim 15, whereinsaid promoter is an inducible promoter.
 19. The vector of claim 15,wherein said promoter is a tissue specific promoter.
 20. A host cellcomprising the nucleic acid of claim
 15. 21. A plant cell that comprisesthe nucleic acid of claim
 15. 22. A transgenic plant comprising thenucleic acid of claim
 15. 23. The nucleic acid of claim 1, wherein saidsequence encodes a protein comprising amino acid residues 51–55 and 57of SEQ ID NO:
 2. 24. The nucleic acid of claim 1, wherein said sequenceencodes a protein comprising amino acid residues 81, 84, 85, 90 and 91of SEQ ID NO:
 2. 25. The isolated or purified nucleic acid sequence ofclaim 14, further comprising a promoter sequence which controls theexpression of said complementary sequence.
 26. A method for regulatingthe differentiation and the proliferation of a plant cell, comprisingtransforming said plant cell with the polynucleotide sequence ofclaim
 1. 27. A method for regulating the differentiation and theproliferation of a plant cell, comprising transforming said plant cellwith the polynucleotide sequence of claim
 2. 28. A method for regulatingthe differentiation or proliferation of a plant cell, comprisingtransforming said plant cell with the polynucleotide sequence of claim14.